Reactive oxygen species (ROS) are not only harmful by-products of metabolism that need be eliminated to prevent injury, but also important messengers serving in numerous signaling pathways. Acting as a signal, ROS circumvents antioxidant defense by overoxidizing peroxiredoxins (Prxs), the enzymes metabolizing peroxides. However, after the signal is conveyed the antioxidant firewall needs to be restored to prevent oxidative stress. Recently we found that sestrins, a family of proteins whose expression is modulated by p53 tumor suppressor (Hi95 and PA26 genes), are required for regeneration of the over-oxidized Prxs containing cysteine sulfinic acid, thus reestablishing the antioxidant defense. Inhibition of sestrins leads to severe oxidative stress, resulting in apoptosis or premature senescence. We hypothesize that sestrins being critical modulators of both antioxidant defense and ROS-related signaling might determine cell type specific responses to pro-oxidant treatments, and the outcome of signaling, thus representing attractive targets for therapy of ROS-related pathologies. In the proposed program, we are going to determine molecular mechanisms of antioxidant activity of sestrin proteins by studying the protein partners involved in the catalysis of peroxides, the mechanisms of sestrin gene regulation at different levels, the changes in the activity and intracellular localization of sestrins depending on stresses and various physiological signals. We are going to study the roles of sestrin family genes in the modulation of normal physiological signaling, as well as in determining cell fate during stress. We shall study the role of sestrin genes in the cytokine signaling by analyzing the efficiency of signal transduction depending on the levels of expression, intracellular localization, and the proportions of different isoforms of Hi95, PA26 and SesnS. By analyzing the modes of regulation of sestrin genes in response to p53, we will elucidate their role in the protection of the genome against oxidative damage, and their contribution to the p53-mediated control of genetic stability. Roles of sestrins in development and organismic responses will be explored in transgenic and knockdown mouse models. The potential involvement of sestrins in cancerogenesis will be evaluated in a lung cancer model. The results will provide an estimate of the potential value of sestrins as targets for therapeutic interventions.